Sulfation of xanthomonas hydrophilic colloid



States 3,446,796 SULFATION F XANTHOMONAS HYDROPHILIC COLLOID Richard G.Schweiger, San Diego, Calif., assignor to Kelco Company, San Diego,Calif., a corporation of Delaware No Drawing. Filed Nov. 16, 1966, Ser.No. 594,666 Int. Cl. C081) 19/00, 19/02 US. Cl. 260234 16 ClaimsABSTRACT OF THE DISCLOSURE wherein R is a lower alkyl radical and R isselected from the class consisting of hydrogen and a lower alkylradical. The essentially undegraded sulfate esters of a Xanthomonashydrophilic colloid and salts thereof are useful in forming thickenedglues.

This invention relates to a method of preparing undegraded sulfateesters of Xanthomonas hydrophilic colloids. Further, the inventionpertains to essentially undegraded sulfate esters of Xanthomonashydrophilic colloids and salts thereof.

An object of the present invention is to provide a process for producingsulfate esters of Xanthomonas hydrophilic colloids in which thepolymeric structure of the colloid remains relatively undegraded.

A further object of this invention is to provide essentially undegradedsulfate esters of Xanthomonas hydrophilic colloids and salts of suchesters.

Additional objects will appear from a reading of the specification andclaims which follow.

In accord with my invention, I first activate the Xanthomonashydrophilic colloid so that it can be more readily sulfated. Activationis accomplished by acidifying the colloid in an aqueous medium and thendehydrating it prior to reaction. After the colloid has been acidifiedand hydrated, it is precipitated from the aqueous medium by the additionof an organic solvent which is miscible with water but which is not asolvent for the Xanthomonas hydrophilic colloid. Suitable solvents arethe lower alcohols such as methanol, ethanol, propanol, isopropanol,etc., a simple ketone such as acetone, glacial acetic acid, and lowerdialkyl amides, as hereinafter described, such as dimethyl formamide,dimethyl propionamide, diethyl acetamide, and the like. A preferredsolvent for precipitating the material from solution is acetone.

After precipitating the Xanthomonas hydrophilic colloid, it is washedwith one of the aforementioned watermiscible solvents (preferablyacetone) to remove substantially all of the water. If a lower alcohol isused for the above precipitation, it is preferably removed prior atent3,446,796 Patented May 27, 1969 0 I RJLN/ In the above formula R and Rmay be a lower alkyl radical such as, for example, a methyl or ethylradical. In addition, R can be hydrogen. A preferred amide for use in myprocess is dimethyl formamide. Other suitable amides may be employed,however, such as dimethyl acetamide, diethyl acetamide, and dimethylpropionamide. If desired, as set forth previously, the amide may beadded directly to the water solution of Xanthomonas hydrophilic colloidto precipitate the colloid from solution. Such procedure is notgenerally employed because the quantities of amide required to causeprecipitation are relatively large and the procedure is not as effectiveas precipitation with acetone.

The above procedure is designed to remove essentially all of the waterfrom the precipitated colloid and, at the same time, to remove anysubstantial amounts of solvents, such as a lower alcohol, which wouldprovide undesired side reactions during the subsequent sulfationreaction.

The Xanthomonas hydrophilic collids which are sulfated according to myinvention are colloidal materials which are produced by bacteria of thegenus Xanthomonas. Illustrative of such colloidal materials is thehydrophilic colloid produced by the bacterium Xanthomonas campestris.This colloidal material is a polymer containing mannose, glucose,potassium glucuronate and acetyl radicals. The said colloid, which is ahigh molecular weight, exocellular material, may be prepared by thebacterium Xanthomonas campestris, by whole culture fermentation of amedium containing 25% commerical glucose, an organic nitrogen source,dipotassium hydrogen phosphate and appropriate trace elements. Theincubation time is approximately 96 hours at 28 C., aerobic conditions.In preparing the colloid as aforesaid, it is convenient to use cornsteep liquor or distillers dry solubles as an organic nitrogen source.

It is expedient to grow the culture in two intermediate stages topromote vigorous growth of the bacteria. These stages may be carried outin a media having a pH of about 7. In a first stage a transfer from anagar slant to a dilute glucose broth may be made and the bacteriacultured for 24 hours under vigorous agitation and aeration at atemperature of about 30 C. The culture so produced may then be used toinoculate a higher glucose (3%) content broth of larger volume in asecond intermediate stage. In this stage the reaction may be permittedto continue for 24 hours under the same conditions as the first stage.The culture so acclimated for use with glucose by the aforementionedfirst and second stages is then added to the final glucose-containingfermentation medium.

In the aforesaid method of preparation of Xanthomonas campesrrishydrophilic colloid, a loopful of organism from the agar slant isadequate for the first stage comprising 200 milliliters of the saidglucose media. In the second stage that material resulting from thefirst stage may be used together with 9 times its volume of a 3% glucosemedia. In the final stage the material produced in the second stage maybe admixed with 19 times its volume of the final media. A good finalmedia may contain 3% glucose, 0.5 distillers dry soluble, 0.5%dipotassium phosphate, 0.1% magnesium sulphate having 7 molecules ofwater of crystallization and water. The reaction in the final stage maybe satisfactorily carried out for 96 hours at 30 C. with vigorousagitation and aeration. The resulting Xanthomonas campestris colloidalmaterial can be recovered by precipitation in methanol of the clarifiedmixture from the fermentation. This resulting material may also bedesignated as pseudoplastic, heteropolysaccharide hydrophilic colloidproduced by the bacterial species Xanthomonas campestris.

Other Xanthomonas hydrophilic colloids may be prepared by repeating theabove procedure for producing a Xanthomonas campestris hydrophiliccolloid by substituting known Xanthomonas bacteria or organisms, i.e.,Xanthomonas cwrotac, Xanthomonas incmzae, Xanthomonas liegoniae, andXanthomonas malvacearum, for the bacterium, Xanthomonas campestris.

As stated previously, the Xanthomonas hydrophilic colloid employed as areactant in my process must first be suitably activated. Activation mayconveniently be accomplished in conjunction with the process forpreparing the Xanthomonas hydrophilic colloid by directly acidifying thefinal fermentation beer containing the colloid. After acidification ofthe beer by the addition of an acid, such as hydrochloric acid, to a pHof about 2, the acidified hydrated colloid is precipitated anddehydrated through addition of a water miscible organic solvent asdescribed previously. Alternatively, of course, the activated colloidmay be prepared by adding the dried Xanthomonas hydrophilic colloid or asalt thereof to water, thereafter acidifying and then precipitating thecolloid by the addition of a water miscible organic solvent.

Following the activation of the Xanthomonas hydrophilic colloid, thematerial is maintained in a wetted state with the solvent or lowerdialkyl amide employed in the final Washing step. It is quite importantthat the activated colloid not be allowed to dry prior to the succeedingsulfation reaction. Should this occur, the colloid may becomedeactivated to some extent such that it will not react in the desiredmanner during the sulfation reaction.

The wetted and activated Xanthomonas hydrophilic colloid is then reactedwith a complex of sulfur trioxide and a lower dialkyl amide of the typedescribed previously. Preferably, the amide is dimethyl formamide ordimethyl acetamide. The complex is formed by adding sulfur trioxide tothe lower dialkyl amide with cooling of the reaction mixture to maintainthe temperature below about 40 C. Preferably, a molar excess of theamide, such as dimethyl formamide, is employed for reaction with thesulfur trioxide. A suitable excess which I have employed uses 2 moles ofthe amide for each mole of sulfur trioxide. The resulting complex is acrystalline solid which is in a wetted condition due to the excess ofamide generally employed in its formation. The wet crystalline solidmaterial can be maintained under refrigeration, for example, at about 4to 5 C., until ready for use.

The activated Xanthomonas hydrophilic colloid is preferably reacted withthe sulfur trioxide-amide complex in a closed mixer provided with acooling jacket. The presence of moisture is undesirable since water willreact preferentially with the sulfur trioxide-amide complex to formsulfuric acid, which produces degradation of the polymeric structure ofthe colloid. It is for this reason that the reaction is preferablycarried out in a closed system or in an equivalent manner such as by'blanketing the reaction mixture with a dry inert gas.

The sulfation reaction is carried out under agitation to promote an evenreaction rate. Since both the sulfur trioxide-amide complex and theactivated colloid are solids, an excess of amide can be employed ifdesired to facilitate mixing and heat control during the sulfationreaction. Excess amide can, for example, be admixed with the activatedcolloid prior to the sulfation step, can be employed in excess in theformation of the sulfur trioxide-amide complex, or can be simply addedto the reaction mixture along with the sulfur trioxide complex and theactivated colloid.

In conducting the sulfation reaction, I have found that a reactiontemperature of about 0 C. to about 25 C. is suitable, and thatpreferably the reaction temperature is maintained below about 15 C. Thereaction time required for complete esterification is generally fromabout one to several hours, depending upon the selected temperature andthe relative concentrations of the reactants, including diluent andsulfur trioxide-amide complex, in the reaction mixture. In general, Iuse a reaction time of about 1 to about 8 hours and preferably 2 to 3hours.

The product which is obtained by esterification may be neutralized byadding a calculated amount of a suitable base to the reaction mixture.Essentially any base can be employed such as an alkali or alkaline earthmetal hydroxide, carbonate, or bicarbonate, e.g., sodium carbonate,potassium hydroxide, magnesium hydroxide, calcium hydroxide, and thelike. Further, compounds such as ammonium hydroxide, or any of thevarious ammonium compounds, or the various amines such as methyl amine,ethyl amine, propyl amine, and the like can be employed.

Also, the sulfated product may be neutralized by first diluting thereaction mixture with water and then adding a base, as defined above. Ifthe base employed is water soluble, if can conveniently be added in theform of an aqueous solution. The neutralized product can be precipitatedby the addition of a water miscible solvent in which the sulfatedproduct is insoluble. Suitable water miscible solvents are thoseoutlined previously and include, for example, acetone and the loweralcohols such as methanol and ethanol.

It should be understood that the esters prepared according to myinvention are half esters of sulfuric acid. Thus, one of the hydrogenions originally present in the sulfuric acid is still free to react witha base to form salts. The course of my process may be visualized ashaving one of the valencies of sulfuric acid esterified with a hydroxylgroup of the Xanthomonas hydrophilic colloid, while the other hydrogenion may be subsequently substituted by salt formation on the addition ofa suitable base. This is merely a way of visualizing the reaction sincethe sulfation is accomplished through use of the sulfur trioxide-amidecomplex described previously and not through the use of sulfuric aciditself as a reactant. To further illustrate my invention, there arepresented the following examples in which all parts and percentages areby weight unless otherwise indicated:

EXAMPLE I Nine gallons of a fermentation beer containing water and aXanthomonas campestris hydrophilic colloid, as described previously, wasacidified to a pH of 2 by the addition of dilute hydrochloric acid. Thecolloid was precipitated by adding the solution slowly to twice itsvolume of isopropanol after which the precipitated colloid was washedwith methanol. A portion of the wet fiber, which was equivalent to g. ofthe dry colloid, was washed 5 times with dimethyl formamide (DMF). Thiscaused the fiber to swell, making filtration difficult. The materialwhich contained about 400 g. of DMF (assuming essentially all the liquidin the fiber to be DMF) was reacted with 260 g. of a sulfurtrioxide-dimethyl formamide (SO -DMF) complex with the reaction beingcarried out in a Day Mixer while cooling with ice water. The SO DMFcomplex contained 100% excess of DMF, i.e., two moles of DMF for eachmole of S0 The reaction product was dissolved in ice water andneutralized with sodium hydroxide. The sodium salt was precipitated byadding the solution to methanol, was washed with methanol and dried at450 C. The yield of product was 183 g. and the degree of substitution(D.S.) was 0.96. A 1% aqueous solution of the product had a viscosity of31.1 cps.; a 1% aqueous solution of the product which had beenreprecipitated from methanol had a viscosity of 48.7 cps, and a 1%aqueous solution of the reprecipitated and dialyzed product had aviscosity of 201 cps.

In the above example and those following, the sulfated product wasreprecipitated from methanol or acetone before determination of the D8.The BS. was determined by adding an aliquot of the sodium salt of thesulfated Xanthomonas hydrophilic colloid to a 10% aqueous solution ofhydrogen chloride. The mixture was heated overnight, e.g., 15-20 hours,at reflux. The free sulfuric acid which was released was then determinedgravirnetrically by adding barium chloride to precipitate the sulfateion as barium sulfate which was collected and weighed. This is astandard analytical procedure.

The viscosities of aqueous solutions of my sulfated products, asreported in the previous example and those following were measured at 25C. using a Brookfield Viscometer, Model LVF, using the appropriatespindle rotating at 60 r.p.m.

EXAMPLE II A 100 gram portion (dry basis) of activated Xanothomonashydrophilic colloid prepared directly from the final fermentation beer,as described in Example I, was washed 3 times with methanol and dried ina warm air stream. It was then mixed with 530 cc. of DMF, cooled at 7 C.for one hour, and then sulfated using the sulfation complex and generalreaction conditions described in Example I. After a reaction time of 2hours, metha- 1101 was added and the separated solids were squeezed outon a Biichner funnel, resuspended in alcohol, and filtered ofli again.This material was dissolved in ice water, neutralized with sodiumhydroxide and precipitated with methanol. After drying in warm airstream, the yield was 75 g. of product. The D5. of the product was 0.1,and the viscosities of 1% aqueous solutions of the product were 2580cps. (as prepared), 4,110 cps. (reprecipitated from methanol), and 4,360cps. (reprecipitated and dialyzed).

As shown by the above example, drying of the activated colloid prior tosulfation resulted in a great reduction in the D8. of the product. Thisdemonstrates the importance of maintaining the activated colloid in awetted state prior to the sulfation reaction so as to preventdeactivation of the colloid with a resultant decrease in the D8. of theproduct.

EXAMPLE III Activated Xanthomonas campestris hydrophilic colloid wasprepared in the manner described in Example I. The precipitated colloidwas washed once with isopropanol, and then washed 3 times with acetone.The wet fiber was then mixed with DMF (500 g. DMF/100 g. of colloid-drybasis) for 30 minutes in the Day Mixer while cooling with ice water. Asulfation complex, as described in Example 1, was added and mixing wascontinued for 3 hours. Solids were then precipitated by adding a mixtureof acetone and methanol to the mix. The precipitated' solids werefiltered off and Washed with acetonemethanol mixture. The washed productwas dissolved in water and neutralized with sodium hydroxide. The sodiumsalt was then precipitated through addition to methanol and theprecipitate was dried. The data and results of several test runs carriedout in the above described manner are set forth in the following table.In each of the test runs, 100 grams of activated colloid were employedWhile the amount of the SO -DMF sulfation complex was varied from run torun.

TABLE Grams of S Yield of sulfated 1% viscosity DMF complex product(grams) (cps.) D.S

In the above table, the grams of sulfation complex including 100% excessof DMF, or two moles of DMF for each mole of S0 are set forth incolumn 1. The resulting yields of the sulfate ester of the Xanthomonascampestris hydrophilic colloid are set forth in column 2, and theviscosities of the products in a 1% aqueous solution at 25 C.(Brookfield Viscometer, Model LVF) are set forth in column 3. The degreeof substitution (D.S.) after dialysis is set forth in column 4.

As shown in the above table, the D5. of the products increased as thequantity of the SO -DMF sulfation complex was increased with respect tothe amount of activated Xanthomonas hydrophilic colloid.

EXAMPLE 1V Six liters of a fermentation beer containing Xanthomonascampeslris hydrophilic colloid (as described in Example I) was adjustedto a pH of 2 by the addition of concentrated hydrochloric .acid andpoured slowly and with stirring into 16 liters of glacial acetic acid.Four more liters of acetic acid and one liter of acetone were added, andthe resulting solids were removed and washed 4 times with acetic acid,While squeezing out each time on a Biichner funnel. Eighty-five grams ofactivated Xanthomonas hydrophilic colloid (dry basis) were recovered.The wet fiber was mixed with 340 cc. of DMF, cooled overnight at 5 C.,and reacted with 340 g. of SO -DMF complex (prepared as in Example I)for 2.5 hours. The reaction product was washed twice with a mixture ofacetone and methanol, dissolved in ice water and neutralized with sodiumhydroxide. The sodium salt Was precipitated by addition of the solutionto methanol and dried at 45 C. in the presence of an air stream. Theyield of sulfated product was 111 grams, and its D.S. was 0.98. Itsviscosity in a 1% aqueous solution was 1,104 cps.

As shown by the foregoing examples, my invention provides sulfate estersof essentially undegraded Xanthomonas hydrophilic coloids and the saltsthereof. The viscosities of the materials prepared according to myinvention are generally in excess of about 40 cps. and afterpurification, i.e., salt removal, are in excess of about 200 cps. in a1% aqueous solution. The viscosities referred to are measured at 25 C.using .a Model LVF Brookfield Viscometer. These viscosities clearly showthat the materials are colloidal in nature and contain the essentiallyundegraded structure of the Xanthomonas hydrophilic colloid. The D8. ofthe sulfated products generally ranges up to about 1 depending on thequantity of the sulfation complex employed.

Although I have demonstrated my invention by reference to sulfate estersof Xanthomonas campestris hydrophilic colloid, it should be understoodthat relatively undegraded sulfate esters of other Xanthomonashydrophilic colloids such as those produced by the bacteria Xanthomonasmalvacearum, Xanthomonas carotae, Xanthomonas begoniae, and Xanthomonasincanae, are part of my invention and may be prepared by the foregoingprocedure. Also included in my invention are the salts of essentiallyundegraded sulfate esters of such other Xanthomonas hydrophiliccolloids.

My novel products can be be employed as viscosity and emulsionstabilizers in certain products which contain proteins. To illustrate,it has been found that my products can be employed for preparingthickened glues.

A glue was prepared by adding 15 parts of dried bone glue (CX Bone Glue,Armour and Company, Adhesive Division, 1355 West 31st Street, Chicago,111.), to parts of water. The mixture was heated to solubilize the boneglue and was then cooled. The resulting glue had a viscosity at 45 C. of12 cps. and a viscosity at 250 C. of 18 cps.

A further glue was prepared by adding 13.5 parts of CX Bone Glue, and1:5 parts of the sodium salt of an essentially undegraded sulfate esterof Xanthomonas campestris hydrophilic colloid having a D8. of 0.52 to105 parts of water. After heating to solubilize the ingredients andcooling, the resulting glue was found to have a viscosity of 2,350 cps.at 45 C and 16,000 cps. at 25 C. A further glue was prepared by adding0.5 parts of CX Bone Glue and 1.5 parts of the sodium salt of an essentially undegraded sulfate ester of Xanthomonas campestris hydrophiliccolloid having a D5. of 0.71 to 105 parts of water. The resulting gluehad a viscosity at 45 C. of 1280 cps. and a viscosity at 25 C. of 8,000cps.

As shown by the above data, those glues which contained an essentiallyundegraded sulfate ester of a Xanthomonas campestris hydrophilic cooloidprepared according to my invention had much higher viscosities than aglue containing only bone glue and water. The thickened glues containingan essentially undegraded ester of a Xanthomonas hydrophilic colloidmay, for example, be employed on a vertical surface where other glueswould have a tendency to run and give uneven covering and non-uniformadhesion.

In the foregoing description, I have referred to various temperatures,times, concentrations and the like. These references to specificconditions have been solely for purposes of illustration. Thus, I desirethat my invention be limited only by the lawful scope of the appendedclaims.

What I claim is:

1. An essentially undegraded sulfate ester of a Xanthomonas hydrophiliccolloid and the base neutralized salts thereof.

2. The product of claim 1 wherein said colloid is a Xanthomonascampestris hydrophilic colloid.

3. The product of claim 1, wherein said colloid is a Xanthomonasmalvacearum hydrophilic colloid.

4. The product of claim 1, wherein said Xanthomonas hydrophilic colloidhas a degree of substitution ranging up to about 1.

5. The product of claim 4, wherein said salt is selected from the groupconsisting of alkali metal, alkaline earth metal, amomnium and aminesalts.

6. Process for preparing an essentially undegraded sulfate ester of aXanthomonas hydrophilic colloid, said process comprising acidifying aXanthomonas hydrophilic colloid in aqueous medium, precipitating saidacidified colloid, removing substantially all of the water from saidprecipitated colloid while maintaining said colloid in a wetted state,and reacting said wetted colloid with a sulfur trioxide-dialkyl amidesulfation complex in which the amide has a formula wherein R is a loweralkyl radical and R is selected from the class consisting of hydrogenand a lower alkyl radical.

7. The process of claim 6, wherein said amide is present in excess insaid sulfation complex.

8. The process of claim 6, wherein said acidified Xanthomonashydrophilic colloid is precipitated from an aqueous medium by theaddition thereto of a watermiscible organic solvent which is not asolvent for said acidified Xanthomonas hydrophilic colloid.

9. The process of claim 8, wherein said water-miscible organic solventis a lower alcohol.

10. The process of claim 8, wherein said water-miscible organic solventis acetone.

.11. The process of claim 6, wherein amide is dimethyl formamide.

112. The process of claim 6, wherein said sulfation reaction is carriedout at a temperature from about 0 C. to about 25 C.

13. The process of claim 12, wherein said sulfation reaction is carriedout at a temperature below about 15 C.

14. The process of claim 6, including the additional step ofneutralizing said essentially undegraded sulfate ester of a Xanthomonashydrophilic colloid.

15. The process of claim 6, wherein said Xanthomonas hydrophilic colloidis prepared by the bacterium Xanthomonas campestris.

16. The process of claim 6, wherein said Xanthomonas hydrophilic colloidis prepared by the bacterium Xanthomonas malvacearum.

References Cited UNITED STATES PATENTS 3,020,206 2/1962 Patton et al.260--209 3,163,602 12/ 1964 Lindblom et al 260-209 3,200,110 8/1965Gollin et al 260--234 3,232,929 2/ 1966 McNeely et al. 260-209 3,256,2716/ 1966 Schweiger 260-234 LEWIS GOTTS, Primary Examiner.

I. R. BROWN, Assistant Examiner.

US. Cl. X.R.

